Prevalence of SARS‐CoV‐2 Antibodies in Kosovo‐Wide Population‐Based Seroepidemiological Study

ABSTRACT Background Seroprevalence studies have proven to be an important tool in tracking the progression of the coronavirus disease 2019 (COVID‐19) pandemic. The aim of this study was to measure the seroprevalence of antibodies to severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) in the general population of Kosovo by gender, age group and region and among asymptomatic people. Method The Institute of Public Health of Kosovo conducted a cross‐sectional population‐based survey, aligned with the protocols of the WHO Unity Studies, from the beginning of May to the end of June 2021. Results The survey covered a total of 2204 people with a response rate of 91.8% (41.9% [923] males and 51.2% [1281] females). In May to June 2021, the prevalence of antibodies in the overall population (IgG antibodies ≥ 1.1) was 37.0%. Seroprevalence was 34.4% in men and 38.9% in women (p < 0.05), with the highest percentage (48.7%) found in the 60–69 years' age group. The overall prevalence of acute IgM antibodies (IgM ≥ 1.1) was 1% (95% CI: 0.7%–1.5%), with no significant difference between genders and the highest prevalence among participants of 60–69 years of age (1.6%; 95% CI: 0.7%–3.6%). Conclusion A high prevalence of antibodies against SARS‐CoV‐2 was found in Kosovo before the start of the vaccination campaign. However, the results of the survey suggested that, by the end of June 2021, a desirable level of protection from the SARS‐CoV‐2 virus had not been reached.

exacerbated by the fragility of its health systems in facing this global challenge.
The first cases of COVID-19 were reported in Kosovo on 13 March 2020, involving a 77-year-old man from Vitia who had recently returned from Italy and an Italian woman in her early 20s [7].In response to the epidemiological situation, the health authorities in Kosovo declared a public health emergency and introduced public health and social measures, including closure of the schools and restrictions on international travel (including quarantine and testing) and movement up to 31 May 2020.
Between 13 March 2020 and 31 June 2021, 107,768 cases of COVID-19 were confirmed in Kosovo, including 2258 deaths, according to an analysis conducted by the Institute of Public Health of Kosovo and the WHO [8].The COVID-19 vaccination programme started on 29 March 2021 with the rollout of vaccinations for health-care workers and people > 65 years, but mass vaccination started on 15 June 2021 [9].Between 29 March and 15 June 2021, 99,000 doses of vaccine were administered [10], resulting in the vaccination of around 5% of population.Initially, the necessary quantities of vaccines were secured: 1.2 million vaccines received through a bilateral contract from Pfizer, donation of vaccines for 20% of the population received through COVAX and other donations.
Seroprevalence studies have proven to be an important tool for monitoring the progression of the COVID-19 pandemic and providing estimates of the true burden of disease.Despite a significant number of seroprevalence studies having been conducted globally [11], there is a paucity of seroprevalence studies after several waves of the pandemic, and before mass vaccination, in Central and Eastern Europe [12,13], especially for the low-and middle-income economies of the WHO European Region.
In 2021, 1 year into the pandemic, the Institute of Public Health of Kosovo conducted a Kosovo-wide seroprevalence study.The aim was to gain an understanding of the true population exposure to the virus prior to mass vaccination, as well as the proportion of asymptomatic infections in the population, and to inform public health strategies to manage emergencies with infective diseases.

| Methods
The Institute of Public Health of Kosovo conducted a crosssectional population-based seroprevalence survey, aligned with the protocols of the WHO Unity Studies, in Kosovo, from the beginning of May to the end of June 2021 regardless of waves, with technical support from the WHO [14].During this period in Kosovo, 2560 positive cases of COVID-19 were reported, with 84 deaths.

| Study Design and Population Recruitment
The survey was carried out using multistage, age-stratified population sampling.During the first stage, the population was stratified according to the 34 municipalities of Kosovo; during the second stage, each health region was stratified by age group (2-9, 10-19, 20-29, 30-39, 40-49, 50-59, 60-69 and 70+ years).[6].The participants were selected from the households sampling list generated randomly by the KAS.One individual per household (defined as a group of people-two or more-living in the same residence) was invited to participate in the serosurvey, selected using Kish method.All individuals identified for recruitment, irrespective of age and acute or prior SARS-CoV-2 infection, were included in the study.Refusal to give informed consent and contraindication to venipuncture were exclusion criteria.

| Data Collection and Laboratory Testing
The survey was conducted in seven sites: the Institute of Public Health and six regional centres of public health.
Informed consent was obtained from all individuals willing to participate in the investigation prior to the performance of any procedure.Consent for the participation of children (< 18 years old) was obtained from the parent/caregiver.Each participant was randomly recruited and requested to complete an epidemiological questionnaire, covering demographic, clinical and exposure-related information.Blood samples were collected from adults and children (5 and 2.5 mL, respectively) through venipuncture by trained staff and transported at 4.0°C to the Department of Microbiology of the Institute of Public Health, where they were aliquoted and stored at −80°C.
In addition, nasopharyngeal swabs were collected for testing for SARS-CoV-2 infection by real-time PCR (RT-PCR).All laboratory investigations were conducted at the Department of Microbiology.
Serum samples were analysed for the presence of specific IgM and IgG antibodies against SARS-CoV-

| Ethical Considerations
The study protocol was approved by the Doctors' Chamber Ethical Committee in Kosovo (reference numbers 20/2020 and 24/2021) and the WHO Research Ethics Review Committee (protocol ID: CERC.0013E).
The data analysis was conducted in Epi Info, Version 3.5.1,developed by the Centers for Disease Control and Prevention, Atlanta, Georgia, USA, and the prevalence rates with corresponding 95% CIs were estimated.The results were considered statistically significant if p < 0.05.
IgM seropositivity was higher among participants over 20 years old, mainly in the region of Gjilan where it was recorded as 3.5% (95% Cl: 1.8-6.9)(Table 2).
Participants were tested for SARS-CoV-2 by RT-PCR (see Table S1).At the time of sampling, the overall PCR positivity was 0.0 (95% Cl: 0.0-0.3).There was only one positive case of women aged 60-69 years from the region of Prizren.3).

| Discussion
This was the first Kosovo-wide cross-sectional SARS-CoV-2 seroprevalence investigation to be carried out.By May-June 2021, 37.0% (95% CI: 35.0-39.1) of the population had been exposed to SARS-CoV-2.The seroprevalence varied by geography, gender and age group, which was consistent with the general picture from the broader surveillance system.Kosovo is a small country with 10,887 km 2 with a population density of 177.4 inhabitants per km 2 [6].The high prevalence in Prizren compared to other regions of Kosovo can be attributed to its proximity to Albania.
In Albania, seroprevalence was 59.4% during July to August 2021 [16].In one systematic review in 2021, Ya'qoub et al. [17] describe sex and gender differences in a lot of studies.These differences are likely attributed to a combination of factors like hormonal differences, immune response and differences in attitudes and behaviors.
Several large seroprevalence studies had been conducted elsewhere in the WHO European Region with varying results [18].In their systematic review, Bergeri et al. showed that, by June 2021, the overall seroprevalence in low-and middle-income economies in the WHO European Region was 48.7% (47.7%-49.7%),compared to 22.4% (21.1%-23.8%) in July 2020 [16].A nationwide study of the general population in Croatia, performed after the first (May-July 2020) and second (December 2020-February 2021) waves of the pandemic, found a significant difference in the overall seroprevalence rate over time (IgG 2.2%-25.1%)[13].
Initially, surveillance in Kosovo focused primarily on patients and contacts with symptoms, with more than half of the confirmed cases showing symptoms and the remaining cases being asymptomatic.In addition, the role of pre-symptomatic, asymptomatic or subclinical infections in the human-tohuman transmission of SARS-CoV-2 virus is not well understood.With a novel coronavirus, initial seroprevalence in the population is assumed to be negligible.Therefore, the surveillance of antibody seropositivity in a population could allow for inferences about the extent of infection and its cumulative incidence in the population.For the survey under discussion, a standardized WHO protocol was used [14].This enabled the systematic collection and rapid sharing of epidemiological exposure data and biological samples in a format that could easily be aggregated, tabulated and analysed across many different settings globally.These timely estimates of SARS-CoV-2 and information about its severity and attack rates made it possible to inform public health responses and policy decisions, which is particularly important in the context of a novel respiratory pathogen, such as SARS-CoV-2.Limitation of this study was only that The COVID-19 vaccination programme started on 29 March 2021 with the rollout of vaccinations for health-care workers and people > 65 years, but mass vaccination started on 15 June 2021 but the exclusion criterion from study was if somebody was vaccinated.Such studies have been performed in many health systems.Some of them have been limited to specific geographical areas or carried out on non-representative samples of the population, but surveys designed as national or equivalent representative surveys have found substantial geographical variability with higher seroprevalence in more densely populated areas [22][23][24][25][26][27][28][29][30][31][32].

| Conclusions
This serological survey demonstrated that SARS-CoV-2 was likely more widespread in Kosovo than indicated by the number of cases reported in June 2021.However, its findings showed that the population of Kosovo had not reached a suitable level of protection from the SARS-CoV-2 virus in the beginning of May to the end of June 2021.Regular seroprevalence studies are required to monitor population seroprevalence, assess risk factors and inform targeted public health policies.

FIGURE 1 |
FIGURE 1| Geographical distribution of SARS-CoV-2 seroprevalence in Kosovo, May-June 2021.The designations employed and the presentation of the material in this publication do not imply the expression of any opinion whatsoever on the part of WHO concerning the legal status of any country, territory, city or area or of its authorities or concerning the delimitation of its frontiers or boundaries.All references to Kosovo in this document should be understood to be in the context of the United Nations Security Council Resolution 1244 (1999).

TABLE 2 |
Prevalence of SARS-CoV2 antibodies (IgG and IgM) by sex, age group and region.Prevalence of IgG ≥ 1.1 Prevalence of IgM ≥ 1.1